The Receptor-Binding Domain of Influenza Virus Hemagglutinin Produced in
            <i>Escherichia coli</i>
            Folds into Its Native, Immunogenic Structure

c Influenza A virus (IAV) remains an important human pathogen largely because of antigenic drift, the rapid emergence of antibody escape mutants that precludes durable vaccination. The most potent neutralizing antibodies interact with cognate epitopes in the globular "head" domain of hemagglutinin (HA), a homotrimeric glycoprotein. The H1 HA possesses five distinct regions defined by a large number of mouse monoclonal antibodies (MAbs), i.e., Ca1, Ca2, Cb, Sa, and Sb. Ca1-Ca2 sites require HA trimerization to attain full antigenicity, consistent with their locations on opposite sides of the trimer interface. Here, we show that full antigenicity of Cb and Sa sites also requires HA trimerization, as revealed by immunofluorescence microscopy of IAVinfected cells and biochemically by pulse-chase radiolabeling experiments. Surprisingly, epitope antigenicity acquired by HA trimerization persists following acid triggering of the globular domains dissociation and even after proteolytic release of monomeric heads from acid-treated HA. Thus, the requirement for HA trimerization by trimer-specific MAbs mapping to the Ca, Cb, and Sa sites is not dependent upon the bridging of adjacent monomers in the native HA trimer. Rather, complete antigenicity of HA (and, by inference, immunogenicity) requires a final folding step that accompanies its trimerization. Once this conformational change occurs, HA trimers themselves would not necessarily be required to induce a highly diverse neutralizing response to epitopes in the globular domain.T he influenza A virus (IAV) hemagglutinin (HA) glycoprotein attaches virions to target cells by binding terminal sialic acid residues on cell surface glycans (1, 2). As a prototypical homotrimeric type I integral membrane protein, HA is synthesized in the endoplasmic reticulum (ER) of infected cells and transported through the Golgi complex (GC) to the plasma membrane (PM), where it is incorporated into budding virions. A variable number (depending on the strain) of N-linked oligosaccharides are added cotranslationally as HA is extruded into the ER through the translocon and subsequently trimmed and modified extensively during HA transport to the cell surface. In addition, the cytoplasmic COOH terminus of HA is palmitoylated during its intracellular transport, likely in an early GC compartment (3).HA folding begins cotranslationally, as shown by the acquisition of intrachain disulfide bonds and by the binding of monoclonal antibodies (MAbs) specific for discontinuous epitopes within HA to nascent chains (4). Initial folding of HA monomers is likely completed shortly after chain termination (within 1 or 2 min), whereas HA trimerization occurs with a half-life of ϳ5 min (5, 6). The localization of the later process appears to be strain (and perhaps subtype) specific, with prototype H2 (7) and H3 (8) HAs trimerizing in the ER while the A/Puerto Rico/8/34 (PR8) H1 HA trimerizes in either the ER-Golgi intermediate compartment or the cis-GC (3, 6).HA is of great medical importance by virtue of its recog...

Section: Discussionmentioning

confidence: 63%

Influenza A Virus Hemagglutinin Trimerization Completes Monomer Folding and Antigenicity

Magadán

Khurana

Das

et al. 2013

“…3A). The lack of carbohydrates on the bacterially expressed HA1 does not affect the folding and antigenic integrity; almost all of the conventional HA antigenic sites, Sa, Sb, Ca1, Ca2, and Cb, which have been classically characterized using polyclonal mouse antisera (37,38), have good structural integrity and align well with the bacterially expressed and refolded HA1 and full-length HAs from the H1 subtype (4,36) (Fig. 3A).…”

Section: Resultsmentioning

confidence: 98%

“…Eukaryotic expression systems are widely used for the preparation of recombinant HAs, as these proteins have glycans on their N-linked glycosylation sites. However, it has been previously reported that HA1 can be recombinantly produced and refolded from E. coli inclusion bodies with similar biophysical properties to HA produced in insect cells (36). Moreover, E. coli-expressed HA1 has been shown to elicit a protective immune response in ferrets (48), suggesting that a protective antibody response can be generated despite the lack of glycan shielding on the surface of HA, at least in the case of the 2009 H1 pandemic strain.…”

Section: Discussionmentioning

confidence: 99%

Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site

Hong

Lee

Hoffman

et al. 2013

145

165

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.

“…These diversified Bcell/Ab epitopes include three previously characterized B-cell/Ab epitopes-Sa, Sb, and Ca2-defined by Caton et al by grouping mutants of the influenza virus A/PR/8/34 virus based on their antigenic reactivity to panels of neutralizing monoclonal antibodies (11,12). These epitopes represent distinct neutralizing antigenic regions which have previously been reported to bind to human sera from patients infected with prepandemic H1N1 (24). Thus, this diversifying selection analysis of prepandemic H1N1 HA has identified a subset of experimentally defined H1 HA Bcell/Ab epitopes that appear to be especially important for protective immunity in humans, including the Sa, Sb, and Ca2 Caton epitopes.…”

Section: Resultsmentioning

confidence: 99%

Diversifying Selection Analysis Predicts Antigenic Evolution of 2009 Pandemic H1N1 Influenza A Virus in Humans

Lee

Das

Wang

et al. 2015

Although a large number of immune epitopes have been identified in the influenza A virus (IAV) hemagglutinin (HA) protein using various experimental systems, it is unclear which are involved in protective immunity to natural infection in humans. We developed a data mining approach analyzing natural H1N1 human isolates to identify HA protein regions that may be targeted by the human immune system and can predict the evolution of IAV. We identified 16 amino acid sites experiencing diversifying selection during the evolution of prepandemic seasonal H1N1 strains and found that 11 sites were located in experimentally de- IMPORTANCEThe WHO estimates that approximately 5 to 10% of adults and 20 to 30% of children in the world are infected by influenza virus each year. While an adaptive immune response helps eliminate the virus following acute infection, the virus rapidly evolves to evade the established protective memory immune response, thus allowing for the regular seasonal cycles of influenza virus infection. The analytical approach described here, which combines an analysis of diversifying selection with an integration of immune epitope data, has allowed us to identify antigenic regions that contribute to protective immunity and are therefore the key targets of immune evasion by the virus. This information can be used to determine when sequence variations in seasonal influenza virus strains have affected regions responsible for protective immunity in order to decide when new vaccine formulations are warranted.